combined-recombinase-polymerase-amplification-crisprcas12a-assay-for

Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting <em>Fusarium oxysporum</em> f. sp. <em>cubense</em> Tropical Race 4

Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most ...

Stellenbosch University

Banana is an important food crop and source of income in Africa. Sustainable production of banana, however, is at risk because of pests and diseases such as Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). Foc can be disseminated from infested to disease-free fields in plant material, water and soil. Early detection of Foc using DNA technologies is thus required to accurately identify the fungus and prevent its further dissemination with plants, soil and water. In this study, quantitative (q)PCR assays were developed for the detection of Foc Lineage VI strains found in central and eastern Africa (Foc races 1 and 2), Foc TR4 (vegetative compatibility groups (VCG) 01213/16) that is present in Mozambique, and Foc STR4 (VCG 0120/15) that occurs in South Africa. A collection of 127 fungal isolates were selected for specificity testing, including endophytic Fusarium isolates from banana pseudostems, non-pathogenic F. oxysporum strains and Foc isolates representing the 24 VCGs in Foc. Primer sets that proved to be specific to Foc Lineage VI, Foc TR4 and Foc STR4 were used to produce standard curves for absolute quantification, and the qPCR assays were evaluated based on the quality of standard curves, repeatability and reproducibility, and limits of quantification (LOQ) and detection (LOD). The qPCR assays for Foc Lineage VI, TR4 and STR4 were repeatable and reproducible, with LOQ values of 10-3-10-4 ng/μL and a LOD of 10-4-10-5 ng/μL. The quantitative detection of Foc strains in Africa could reduce the time and improve the accuracy for identifying the Fusarium wilt pathogen from plants, water and soil on the continent.

Quantitative detection of economically important Fusarium oxysporum f. sp. cubense strains in Africa in plants, soil and water.

Fusarium wilt, caused by the soil-borne fungus  f. sp.  (Foc) race 1, is a major disease of bananas in East Africa. Triploid East African Highland (Matooke) bananas are resistant to Foc race 1, but the response of diploid (Mchare and Muraru) bananas to the fungus is largely unknown. A breeding project was initiated in 2014 to increase crop yield and improve disease and pest resistance of diploid and triploid East African Highland bananas. In this study, eight Mchare cultivars were evaluated for resistance to Foc race 1 in the field in Arusha, Tanzania. In addition, the same eight Mchare cultivars, as well as eight Muraru cultivars, 27 Mchare hybrids, 60 Matooke hybrids and 19 NARITA hybrids were also screened in pot trials. The diploid Mchare and Muraru cultivars were susceptible to Foc race 1, whereas the responses of Mchare, NARITAs and Matooke hybrids ranged from susceptible to resistant. The Mchare and Matooke hybrids resistant to Foc race 1 can potentially replace susceptible cultivars in production areas severely affected by the fungus. Some newly bred Matooke hybrids became susceptible following conventional breeding, suggesting that new hybrids need to be screened for resistance to all Foc variants.

Evaluation of Mchare and Matooke Bananas for Resistance to  f. sp.  Race 1.

Nontuberculous mycobacteria (NTM) infections present a global health challenge. This study describes unidentified mycobacteria species from extrapulmonary sites, using advanced identification and sequencing techniques.

Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures.

The soilborne pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is currently devastating banana production worldwide. Once introduced, it is not possible to eradicate the pathogen from soils where it can survive for decades. The only management option available then is to replace Foc TR4-susceptible with -resistant varieties. Timely detection of the pathogen, however, is an important strategy to prevent the introduction of Foc TR4 into new areas and prevent its spread from infested sites. In this study, a single-tube detection technique was developed by combining recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology (RPA-Cas12a) for detection of Foc TR4. The RPA-Cas12a assay was conducted isothermally, had a sensitivity of up to 10 fg target DNA and did not cross react with any of the 76 non-target isolates included in the specificity testing. The RPA-Cas12a assay detected Foc TR4 from naturally infected banana samples collected in the field and visualization was possible with the naked eye under LED blue light transillumination. The method can be integrated with inexpensive fluorescent or electronic detection devices to accelerate Foc TR4 in-field detection and, thereby, fast-track disease containment strategies.

Article	Year
Quantitative detection of economically important Fusarium oxysporum f. sp. cubense strains in Africa in plants, soil and water. PloS one\| PubMed ID: 32687514	2020
Evaluation of Mchare and Matooke Bananas for Resistance to f. sp. Race 1. Plants (Basel, Switzerland)\| PubMed ID: 32842551	2020
Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures. IJID regions\| PubMed ID: 39483154	2024
A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4. Scientific reports\| PubMed ID: 39828694	2025

Megan Ceris Matthews

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